Lipoproteins, which are carriers of lipids, cholesterol and triglycerides, are amongst the major components of blood plasma or serum (for brevity hereinafter the term “plasma” will be used but references to “plasma” should be interpreted as references to plasma or serum). The lipoproteins found in blood plasma fall into three main classifications: high density lipoproteins (HDL), low density lipoproteins (LDL) and very low density lipoproteins (VLDL). It is well known that there is a strong relationship between the concentration of lipoproteins in blood plasma and the risk of atherosclerosis (i.e. cardio-vascular desease development). It is also known that the different classes of lipoproteins each play a different role in atherosclerosis. For instance, HDL is regarded as antiatherogenic whereas LDL is known to be highly atherogenic (the cholesterol it carries correlating closely with atheroscleroses development). VLDL is considered to be slightly atherogenic and of more significance in females.
Blood plasma is a complex mixture of a variety of proteins and although methods for separating and directly measuring the concentration of different classes of lipoproteins are known, such methods are complex and expensive. Accordingly, the conventional method of lipoprotein assay widely used in clinical laboratories is an indirect method in which the important LDL concentration is calculated from the measurement of total cholesterol concentration, triglyceride concentration and HDL concentration using the Fridedewald equation:(CH-LDL)=CH−(CH-HDL)−TG/5where CH is the total cholesterol concentration, (CH-LDL) is the cholesterol LDL concentration, (CH-HDL) is the RDL concentration and TG is the triglyceride concession (including free glycerol).
The HDL, CH and TG concentrations must be determined before the LDL concentration can be calculated and it will be appreciated that any errors in the measurement of the HDL, CH and TG concentrations will be compounded in the calculation of the LDL concentration. In addition, the conventional measurement of TG concentration does not discriminate between triglycerides and free glycerol, concentrations which can vary introducing a further error into the calculation of the LDL concentration. Thus, the calculation of LDL concentration inherently includes errors which can be extremely significant, particularly at high triglyceride levels. Such errors are a particular problem in, for instance, monitoring the progress of widely used LDL decreasing treatments (such as diets, medicine etc) in which it is necessary to accurately monitor relatively small decreases in LDL concentration (typically of the order of several percent) whilst triglyceride levels may be changing dramatically.
A further disadvantage of the conventional assay procedure described above is that whilst total CH and TG assays involve straightforward techniques performed on the whole plasma sample, the measurement of he HDL concentration requires a preliminary separation process in which the LDL and VLDL components are removed (by coagulating the LDL plus VDL and removing them by centrifuge).
A yet further disadvantage of the above method is that it provides no measurement of VLDL concentration which must therefore be determined from some different method or process, for instance by calculating the VLDL concentration on the basis of plasma triglyceride and free glycerol content estimated enzymatically.
An alternative method for assaying the total lipoprotein concentration of blood plasma, which is mainly the sum of LDL plus VLDL, is disclosed in two Russian patents, numbers SU1457386 and SU1476384. These relate to the use of a particular organic luminophore, 4-dimethylamino-4′-difluoromethyl-sulphonyl-benzylidene-acetophenone (DMSBA), as a florescent probe. The formula of the probe, identified as K-37, is given below:

The probe K-37 is not luminous in water but is highly luminous in aqueous protein solutions such as blood plasma. In particular, the intensity of the florescence is highly dependent upon the lipoprotein content of the blood plasma and thus K-37 can be used as a probe to discriminate between lipoprotein concentration and the concentration of other proteins that may be present.
Russian patent number SU1457386 describes a method for synthesising K-37 and Russian patent number SU1476384 describes a method of calculating the total CH and TG content of blood plasma from a measurement of the steady state fluorescence of a mixture of a sample of blood plasma and K-37 (with reference to the measured florescence and known concentration of a standard sample). A blood sample to be assayed is diluted using a buffer solution (pH7.4, containing 10 mN Tris-HCL and 2 mN EDTA) and then centrifuged to remove red blood cells and other formed elements. A small amount (10 μl) of a 1 mN standard solution of K-37 is then added to 1 ml of the supernatant solution and the intensity of the resultant florescence is measured using a florescent spectrophotometer, at excitation and observation wavelengths of 440 nm and 550 nm respectively. A formula is disclosed for calculating the overall concentration of CH and TG from the measured florescence of the sample to be assayed and the measured florescence of K-37 in a standard sample of plasma with a known CH and TG concentration at a known dilution. Results for the repetition of the process for a number of different dilutions of the sample to be assayed from 50 fold to 500 fold dilution establishes that the determination of the total concentration of CH and TG is largely independent of the blood dilution.
Thus, use of K-37 as a florescent probe provides a very sensitive assay procedure, so that only very small plasma samples are required, which is relatively simple to perform requiring no separation of protein components. The concentration of CH and TG determined in this way can then be used as a direct indication of hyperlipidemia by comparing the calculated results with normal values.
One shortcoming of the above method is that it does not discriminate between the different classifications of lipoprotein and thus cannot, for instance, be used to monitor small changes in LDL concentration.
It is an object of the present invention to obviate or mitigate the above disadvantages.